Botulinum toxins are among the most deadly biological agents with the capacity of mass destruction. The tripartite toxin consists of a receptor binding domain, a membrane translocation domain and a proteolytic catalytic domain. Botulinum toxins bind specifically to synapses at the neuromuscular junctions by the receptor binding domain, penetrate the nerve cells by the translocation domain, destroy secretary protein assembly by the catalytic domain, and result in paralysis and possible death. No therapeutic drugs are currently available to treat affected individuals. In view of notable recent successes of peptide and recombinant protein therapeutics of anthrax, breast cancer, arthritis, etc., the objective of the proposed project is to develop recombinant protein drugs for botulism. The Specific Aims for the granting period will be first isolating peptides that will not only specifically inhibit the proteolysis but will also rescue damaged nerve cells and, secondly, to develop a targeting and delivery system for such peptides. Peptides that bind the catalytic domain will be initially isolated through combinatorial phage display peptide libraries based on structures of existing inhibitors as well as random search. A high throughput assay of the protease activity in botulinum toxins will then be used to identify inhibitory peptides. Further modifications and expansion of the inhibitory peptide structure will be made to improve the affinity and the specificity of the peptides. Peptides that rescue damaged cells from the toxicity will be selected. Recombinant proteins will then be synthesized that 1) have identical synaptic binding properties as botulinum toxins; 2) contain the intact translocation domain; 3) contain an inactive proteolytic catalytic domain; and 4) fuse with toxin inhibitors, neutralizing proteins, and rescue peptides. The effectiveness of the recombinant proteins in neutralizing and inhibiting botulinum toxin will be examined in vitro and in vivo. Such recombinant proteins are expected to bind to the same target nerve cells as botulinum toxins, to translocate the catalytic domain across the membranes, and eventually to specifically inhibit the degradation of synaptic proteins by the toxins and rescue damaged cells in vivo. The results could be readily applicable to other toxins of the same family and helpful for vaccine development.